Method and means of determining endpoint times in blood clotting tests



July 29, 1969 w. GROSS ET AL 3,453,237

METHOD AND MEANS O1" DETERMINING ENDIOINT TIMES IN BLOOD CLOTTING TESTS 2 Sheets-Sheet 1 Filed April 29, 1965 Y m mm aw TIME SECON S w m w m 5 u @w w w MHWMM w m lzlii Mm .Jp 5 m w I IN 1N o 0 w w HHHHH m R E E S s S a H I [0 0 N If IE E h E M m m I m M. M5

LY m '3 N PE 0 MODIFIED SIGNAL WILLIAM GROSS BYWEL Ll NGTON B. STEWART July 29, 1969 w GROSS ET AL 3,458,287

METHOD AND MEANS OF DETERMINING ENDPOINT TIMES IN BLOOD CLOTTING TESTS Filed April 29. 1965 2 Sheets-$heet 2 INVENTORS AAAIA RUM T M GROSS W I LLIA B WELLI NGTON B. $TEWART EE N N fimux m EEF Kw a U MM a a RNEY United States Patent METHOD AND MEANS OF DETERMINING END- POINT TIMES IN BLOOD CLOTTING TESTS William Gross, Blauvelt, N.Y., and Wellington B. Stewart,

Lexington, Ky., assignors to Medical Laboratory Automation, Inc., a corporation of New York Filed Apr. 29, 1965, Ser. No. 451,885 Int. Cl. GOln 31/14 U.S. Cl. 23-230 9 Claims ABSTRACT OF THE DISCLOSURE Automatic determination of prothrombin time in blood clotting tests by generating a second differential signal from the optical density versus time signal obtained in a photometric apparatus.

This invention relates to blood plasma clotting time determinations and more particularly to a method and means for making such determinations. The invention will be described with particular reference to a prothrombin time determination.

It is a common procedure today to manage and prevent coronary occlusions and other thrombotic states through the use of anti-coagulant drugs. This treatment is employed since it is felt that individuals whose blood has been moderately anti-coagulated are less susceptible to further thrombosis than untreated individuals.

The most commonly used anti-coagulant drugs are of the dicoumarin group. These reduce the clotting ability of blood and consequently patients receiving such therapy must be carefully controlled since if the ability of the blood to clot is reduced too much, the patient is in danger of internal hemorrhage. This eventually would require a halt to the anti-coagulant therapy and the administration of vitamin K an antidote to dicoumarol. On the other hand, if the clotting ability is not reduced sufficiently, the therapy will not be eifective to minimize the recurrence of an occlusion. A test considered significant in estimating clotting ability is the prothrombin time test. As a result of anti-coagulant therapy, this test has become one of the most commonly performed laboratory tests, ranking in number only slightly behind blood sugar and blood urea tests in many laboratories.

The prothrombin time is determined by mixing fixed quantities of blood plasma with thromboplastin-calcium chloride solution and recording the time that elapses before clot formation begins. The prothrombin time of a person having no thrombotic difiiculties is around twelve seconds. The aim of anti-coagulant therapy is to lengthen this time to twenty-five to thirty seconds and then to stabilize the time at that level. If the prothrombin time reaches the range of thirty-five to fifty seconds the aforementioned danger of internal hemorrhage will be present. The drug dosage can usually be regulated by determining prothrombin time once a week or even once every two weeks.

In a small percentage of cases the prothrombin time is also used to investigate disorders of clotting unrelated to anti-coagulant therapy and occasionally as a liver function test. Nevertheless, the bulk of prothrombin time determinations are for regulation of anti-coagulant therapy.

At the present time, prothrombin time is determined generally by either of the following methods. The most extensively practised method may be designated the visual method. In this method the plasma and the reagent are mixed together in a test tube and the reaction visually observed by the technician. A stop watch is started when the mixture is effected, and when he sees formation of a clot begin, the stop watch is halted. The time indicated on the watch is the prothrombin time. It is obvious that, even with a skilled technician, the prothrombin time obtained by this method is subject to inaccuracies since it is dependent on the technicians judgment. This, of course, becomes less reliable as the number of tests increases and tedium takes place. One other method depends on the retention of blood fibrin between two closely spaced electrodes to complete a timer controlled circuit. Another method utilizes photometric techniques and takes advantage of the fact that the optical density of the reaction mixture (plasma and thromboplastin-calcium chloride solution) changes in time, the change being most pronounced when a clot begins to form. However, the disadvantage of this latter method is that the technicians determine the prothrombin time by observation and interpretation of a signal, usually read out on a voltmeter, to detect the beginning of clotting, or by a retrospective study of a recording of the optical density changes.

It is the object of the present invention to provide an improved method and means for determining prothrombin time by photometric techniques.

Prothrombin time is determined according to the present invention by taking the second differential of the optical density versus time curve, detecting when a voltage proportional to this second differential changes sign, and measuring the time interval between the addition of the thromboplastin solution to the plasma and the detection of the change in sign of the second differential signal.

In carrying out the invention, there is provided a photoelectric scanning apparatus into which can be placed a test tube containing plasma-thromboplastin solution, and a circuit responsive to the optical density of the solution that provides a signal at the time clot formation begins in the solution.

More specifically, the electrical circuit provides initially a signal that corresponds to the optical density of the plasma thromboplastin solution, a signal proportional to the first differential of the optical density signal, a signal proportional to the differential of the first differential signal, i.e., the second differential of the optical density signal, and a signal when the second differential signal changes sign. A timer also is provided to record when, after the test is started, the aforesaid signal is received thus providing the prothrombin time.

A feature of the present invention is that the prothrombin time as determined by the present method is independent of the subjective judgment of the technical performing the test.

Another feature of the invention is that the prothrombin time as determined by the present method is relatively independent of fibrinogen level of the plasma.

Other features and advantages of the invention may be gained from the foregoing and from the description of a preferred embodiment thereof which follows.

In the drawings:

FIG. 1 is a curve showing the change in optical density occuring during a prothrombin time determination when oxalated plasma is tested;

FIG. 2 is a similar curve when a citrated plasma is used as the unknown;

FIG. 3 is a curve showing an electrical signal corresponding to the optical curve of FIG. 1;

FIG. 4 is a curve showing an electrical signal corresponding to the second differential of the curve of FIG. 1;

FIG. 5 is a curve showing the signal of FIG. 4 that has been modified to occur only at the instant of clot formation; and

FIG. 6 is a schematic circuit diagram of a preferred means for carrying out the present invention.

In determining prothrombin time, a quantity of fresh blood is collected into an oxalate solution and the plasma is separated therefrom in a centrifuge. A 0.1 ml. sample of the anti-coagulated plasma is then mixed with 0.2 ml. of a thromboplastin-calcium chloride solution and the time measured to the formation of a fibrin clot. The temperature of the oxalated plasma and the thromboplastincalcium chloride solution, as well as the pipettes used in measuring the quantities of solutions used, and the test tubes into which the solutions are placed, are maintained at a temperature of 37 degrees Centigrade, normal blood temperature, to insure an accurate prothrombin time determination since temperature markedly affects the velocity of the reaction.

Instead of mixing the fresh blood with an oxalate solution, it may be anti-coagulated with sodium citrate. This will not adversely affect the determination of prothrombin time by the method and means of the present invention as will be clear from the following specification.

Referring now to the drawings, FIG. 1 shows the characteristic change of optical density of the reagent mixture with respect to time during a prothrombin time determination. It has been demonstrated, and is now well recognized, that the changes in optical density exactly parallel the other physical and chemical events of clot formation. The initial fast rise in optical density occurring soon after mixing of oxalated plasma and thromboplastin-calcium chloride reagent is due to the formation of a white precipitate of calcium oxalate. This precipitation is almost complete within ten to twelve seconds. This early rise in optical density is not seen in citrated plasma, FIG. 2, because calcium citrate does not precipitate. Otherwise the curves of FIG. 1 and FIG. 2 are similar.

It might be well to note here that the optical density of the plasma sample before the addition of the thromboplastin solution may vary depending on the colors of the plasma which in turn is determined by the fat and/or jaundice content of the plasma. This initial optical density, as shown on the curve to the left of the zero time abscissa, does not affect the outcome of the test which according to the present invention depends on the rate of change of the slope of the curve rather than on an absolute value of optical density.

At the time of clotting there is a second increase of optical density which is caused by clot formation, which is actually a polymerization of soluble fibrinogen molecules to more optically dense and insoluble fibrin clot. The slope of this second rise in the curve represents the rate of formation of fibrin from fibrinogen and the total height of this second rise, which requires about twentyfive seconds to be completed in a prothrombin time determination, corresponds to the original fibrinogen level of the plasma sample. The point on the curve which is the desired endpoint of the prothrombin time is exactly the beginning of this second optical density rise due to the formation of fibrin.

A photoelectric cell observing the above reaction produces an electrical signal of exactly the same shape and time relationships as the curve of optical density changes, or what may be considered a mirror image of the curve depending on the circuit arrangement. As will be seen, in the circuit to be described, as the optical density increases, the signal representing the same decreases.

FIGS. 3 and 4 of the drawing show the electrical signal and the second differential corresponding to the optical density curve of FIG. 1. It is obvious from FIG. 4 that when the endpoint of the prothrombin time determination occurs, the electrical signal goes from negative to positive and, therefore, can be measured accurately by suitable instrumentation. It might be well to note here that the endpoint may be indicated by the electrical signal going from a positive to a negative value. In this latter case a circuit arrangement different from the preferred one hereinafter described would be used. FIG. 5 shows a modified signal which is obtained from a circuit that responds to positive voltages only and which is rendered operative only after a fixed period of time, i.e., after a positive voltage caused by formation of the calcium oxalate precipitate.

Reference is now made to FIG. 6 which shows an electrical circuit that has been found satisfactory in carrying out the present invention.

The circuit is connected to an A.C. source by means of plug 20. The lines from the plug to the circuit proper are fused by a fuse 21 and extend through a double pole single throw switch 22. The circuit then extends to terminals Y-Y which lead to the timer circuits which will be described hereinafter. Also, after leaving switch 22 the conductors lead to a transformer 23 which is connected to the full wave rectifier 24. This is a 6X5 tube and it provides a 250 volt DC. potential at conductors 25 and 26. The output of rectifier 24 is filtered by resistor 30 and capacitor 31. A second winding on transformer 23 provides a suitable voltage for the filaments of the various electron tubes used.

Conductor 26 is connected to the circuit start relay CS which is wired in the plate circuit of triode 33, one half of a 6BZ8 tube. The grid of triode 33 is connected to the time delay circuit comprising resistor 34, capacitor 35, contacts SR3, capacitor 36, resistor 37, and contacts SR2. A resistor 40 is provided to limit the current through capacitor 36 when it is initially connected to line 26 by contacts SR2. This time delay circuit is utilized to provide the modified signal of FIG. 5 and its operation will be more fully explained after the remainder of the circuit is described.

The potential provided by conductor 25 is applied to the potentiometer formed by resistors 41 and 42 to give a voltage of volts at point 43. This voltage is applied across the type 6694A photocell 44 and resistor 45. The photocell is arranged to respond to the light emanating from the lamp 46 and passing through the plasma contained in the test tube 47. The lamp 46 is energized preferably by a battery 48 rather than a more conventional power supply in order to avoid line noise. Of course, with suitable noise supression circuits, the lamp could be connected to the A.C. source.

The signal obtained at point 49 is shown in FIG. 3 and is filtered by resistors 50 and 51 and capacitors 52 and 53. If the positions of photocell 44 and resistor 45 are transposed, the signal obtained at point 49 would exactly parallel the change in optical density and the FIG. 3 curve would be similar to the FIG. 1 curve. In this case the endpoint of the prothrombin time determination would be indicated by the second differential signal going from a positive to a negative value.

The signal obtained at point 49 is then applied to the RC circuit 54 which, in effect, gives an output signal at point 55 which is the first differential of the signal at point 49. The first differential signal is next fed to an amplifier 56 (type 6CB6 tube) and then to the second RC circuit 57 which provides a signal that is the second differential (FIG. 4) of the original signal at point 49 except that it has been reversed in sign by the amplifier 56. The second differential signal is next fed to the grid of triode 61, which is the second half of the 6BZ8 tube previously referred to in connection with triode 33.

It was found that the clotting process is extremely smooth and it was therefore necessary to use relatively long time constants in the differentiation circuits 54 and 57. This, however, allowed the signal to be heavily filtered to remove unwanted higher frequency noise which might trigger the circuit spuriously.

If the second differential signal goes negative, the signal is shorted out by the 6AQ5 tube which is wired as a diode 60. However, when a positive second differential signal is transmitted to the grid of triode 61, the plate current will increase to the point where the endpoint relay EP is actuated to indicate that the endpoint of the prothrombin time determination has been reached. If contacts CS1 are separated, a positive signal on the grid of tube 61 will be shorted out by the grid-cathode circuit of the tube.

The timer start and reset circuit includes a Standard Electric Time Company, Model S60 timer 62. One set of contacts 64 of pushbutton switch 63 connects A.C. voltage from the terminals YY to the reset and common terminals of timer 62. The second set of contacts 65 of switch 63 complete a circuit to energize starting relay SR. This relay is of the latching type which each time it is energized moves to the other position where it remains until energized again.

In operation, a sample of anti-coagulated plasma, prepared as before described, is placed in a test tube which is then placed between lamp 46 and photocell 44. Plug 20 is inserted in the AC. service outlet and switch 22 closed. Capacitor 36 begins to charge through contacts SR2 and the voltage across the capacitor continues to rise until it is fully charged. Also, the contacts of starting relay SR are in the condition indicated on the circuit diagram.

At this time the thromboplastin solution is injected into the plasma and the pushbutton 63 simultaneously depressed to start the timer 62. This is effected by contacts 64 resetting the timer to zero and contacts 65 completing a circuit to energize starting relay SR. Energization of the relay SR engages contacts SR1 to start the timer in operation. Contacts SR2 separate to disconnect the capacitor 36 from conductor 26, and contacts SR3 engage to connect the capacitor to the grid of tube 33. The capacitor immediately begins to discharge through resistor 37 for a purpose hereinafter described.

As soon as the thromboplastin solution is injected into the plasma a noise signal is generated by the mixing of the two ingredients. Thereafter, the optical density of the plasma solution undergoes a first rise due to the formation of the calcium oxalate precipitate. This results in a positive second differential signal at point 55, but this has no effect on the energization of triode 61 because contacts CSl are separated to prevent a positive potential being applied to the plate of triode 61.

At a time after the first rise in optical density and after the second differential signal goes negative the charge on capacitor 36 decreases to a point where the bias on the grid of tube 33 is sufficiently low to cut the tube off thereby deenergizing relay CS and engaging contacts CS1. The time constant for this circuit is long enough to insure that relay CS will remain energized until the second differential goes negative. Now, therefore, when the second differential signal next goes positive, tube 61 begins to conduct and energize relay EP. This, as previously mentioned, occurs at the endpoint of the prothrombin time determination. Energization of relay EP causes contacts EPl to engage and complete a circuit for energization of relay SR. This causes contacts SR1 to separate and halt timer 62. The reading of timer 62 therefore gives the prothrombin time. Contacts SR2 engage and contacts SR3 separate to prepare the circuitry for the next prothrombin time determination.

Practically all diagnostic laboratory tests performed on blood plasma to measure clotting ability or clotting deficiency as well as assays for the various specific clotting factors of the blood, such as anti-hemophilic globulin (H.H.G. or Factor VIII), depend on the timing of the beginning of clotting in a reaction tube. Although the present method of spectrophotometric clot detection has been described only in terms of the prothrombin time test, the principle is applicable to all of the other diagnostic tests employing clot timing as their endpoint. The most commonly performed tests using clot timing are the partial thromboplastin time, the prothrombin consumption test, calcium clotting time, the prothrombin and proconvertin (p and p test), the thrombotest, the two stage prothrombin test, the thrombin time, the thromboplastin generation test, the thrombin generation test, and assays for the various clotting factors.

Having thus described the invention, it is to be understood that many changes could be made in the described circuitry without departing from the spirit and scope of the invention, and, therefore, the specification and drawings are to be interpreted in an illustrative rather than a limiting sense.

What is claimed is:

1. The method of determining endpoint time in blood clotting tests which comprises the steps of placing a liquid mixture of plasma and reagent between a light source and a photosensitive device, measuring the change in optical density of the liquid mixture as a function of -time," determining the second differential of the optical density-time curve, and measuring the interval of time between mixing of the plasma and reagent and when the second differential goes from a negative to a positive value.

2. The method of deter-mining endpoint time in blood clotting tests which comprises the steps of placing a liquid mixture of plasma and reagent between a light source and a photosensitive device, producing an electrical signal that is responsive to the change in optical density of the liquid mixture, producing a second differential signal corresponding to the aforesaid signal, and measuring the time interval between mixing of the plasma and reagent and when the second differential signal changes sign.

3. The method of determining endpoint time in blood clotting tests which comprises the steps of placing a liquid mixture of plasma and reagent between a light source and a photosensitive device, producing an electrical signal that is responsive to the change in optical density of the liquid mixture, producing a first differential signal corresponding to the aforesaid electrical signal, producing a second differential signal corresponding to the aforesaid electrical signal, and measuring the time interval between mixing of the plasma and reagent and when the second differential signal changes sign.

4. The method of determining endpoint time in blood clotting tests which comprises the steps of placing a liquid mixture of plasma and reagent between a light source and a photosensitive device, producing an electrical signal that is responsive to the change in optical density of the liquid mixture, producing a second differential signal corresponding to the aforesaid signal, suppressing said second differential signal for a predetermined period of time, and measuring the interval of time between mixing of the plasma and reagent and when the second differential signal changes sign.

5. The method of determining endpoint time in blood clotting tests which comprises the steps of placing a liquid mixture of plasma and reagent between a light source and a photosensitive device, producing an electrical signal that is responsive to the change in optical density of the liquid mixture, producing a first differential signal corresponding to the aforesaid electrical signal, producing a second differential signal corresponding to the aforesaid electrical signal, suppressing said second differential signal for a predetermined period of time, and measuring the time interval between mixing of the plasma and reagent and when the second differential signal changes sign.

6. The method of determining endpoint time in blood clotting tests which comprises the steps of placing a liquid mixture of plasma and reagent between a light source and a photosensitive device, measuring the change in optical density of the liquid mixture as .a function of time, determining the second differential of the optical densitytime curve, and measuring the interval of time between mixing of the plasma and reagent and when the second differential after a predetermined time goes from a negative to a positive value.

7. Apparatus for determining endpoint time in blood clotting tests wherein a liquid mixture of plasma and reagent is placed between a light source and a photosensitive device, said apparatus comprising means for producing an electrical signal that is responsive to the change in optical density, means for producing a second differential electrical signal corresponding to said electrical signal, and means for indicating when said second differential signal changes sign.

8. Apparatus according to claim 7 including a timer, means to start said timer, and means responsive to the indicating means to stop said timer and thereby register prothrombin time.

9. Apparatus according to claim 8 including means to prevent stopping of the timer before the elapse of a predetermined time after starting of the timer.

8 References Cited UNITED STATES PATENTS 2,878,106 3/1959 Malmstadt 23-253 3,267,364 8/1966 Page et al. 23-230 XR 3,307,392 3/1967 Owen et al.

MORRIS O. WOLK, Primary Examiner R. M. REESE, Assistant Examiner US. Cl. X.R. 

